Food Hydrocolloids 19 (2005) 209–220

www.elsevier.com/locate/foodhyd

Influence of environmental stresses on stability of O/W emulsions

containing droplets stabilized by multilayered membranes produced
by a layer-by-layer electrostatic deposition technique
Tomoko Aoki, Eric A. Decker, D. Julian McClements*
Biopolymers and Colloids Research Laboratory, Department of Food Science, University of Massachusetts, Amherst, MA 01003, USA
Received 26 March 2004; revised 19 May 2004; accepted 20 May 2004

Abstract
A three step process, based on electrostatic layer-by-layer deposition, was used to produce oil-in-water emulsions containing submicron
(d32 < 0:3 mm) oil droplets stabilized by sodium dodecyl sulfate (SDS) – chitosan– pectin membranes (100 mM acetic acid, pH 3.0).
First, primary emulsions (5 wt% corn oil, 5 mM SDS) containing anionic droplets (z < 260 mV) stabilized by SDS membranes were
prepared using a high-pressure value homogenizer. Second, secondary emulsions (1 wt% corn oil, 1 mM SDS, 0.024 wt% chitosan)
containing cationic droplets (z < þ59 mV) stabilized by SDS – chitosan membranes were formed by diluting the primary emulsion with
aqueous chitosan solution and agitating to disrupt any flocs. Third, tertiary emulsions (0.2 wt% corn oil, 0.2 mM SDS, 0.0048 wt% chitosan,
0.040 wt% pectin) containing anionic droplets (z < 214 mV) stabilized by SDS – chitosan –pectin membranes were formed by diluting the
secondary emulsion with aqueous pectin solution. The stability of primary, secondary and tertiary emulsions to pH, ionic strength, thermal
processing and freeze – thaw cycling were determined. The droplets in tertiary emulsions had good stability to droplet aggregation over a
wide range of pH values (pH 3.0– 8.0), NaCl concentrations (#500 mM NaCl), thermal treatments (30– 90 8C for 20 min) and freeze – thaw
cycling (2 20 8C for 22 h/30 8C for 2 h). The interfacial engineering technology utilized in this study could lead to the creation of food
emulsions with improved stability to environmental stresses.
q 2004 Elsevier Ltd. All rights reserved.
Keywords: Emulsion stability; Layer-by-layer deposition; SDS; Chitosan; Pectin

1. Introduction Krog & Sparso, 2004; Stauffer, 1999). Each type of

Conventionally, oil-in-water emulsions are created by
homogenizing oil and aqueous phases together in the
presence of one or more emulsifiers (Friberg, Larsson, &
Sjoblom, 2004; McClements, 1999). An emulsifier is a
surface-active molecule that adsorbs to the surface of the
emulsion droplets to form a protective membrane that
prevents them from aggregating (flocculating and/or
coalescing) with one another. In addition, an emulsifier
reduces the oil –water interfacial tension, thereby facilitating
the disruption of emulsion droplets during homogenization
(Walstra, 1993, 2003). A wide variety of different emulsifiers
are available for utilization in the food industry, including
proteins, polysaccharides, phospholipids and small
molecule surfactants (Charlambous & Doxastakis, 1989;
* Corresponding author. Tel.: þ 1-413-545-1019; fax: þ1-413-545-1262.
E-mail address: mcclements@foodsci.umass.edu (D.J. McClements).
0268-005X/$ - see front matter q 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodhyd.2004.05.006
emulsifier varies in its effectiveness at producing small oil
droplets during homogenization, and its ability to prevent
droplet aggregation under different environmental stresses,
such as pH, ionic strength, heating, and freezing (Dickinson,
2003; Garti & Reichman, 1993; McClements, 1999).
Food emulsifiers also differ in their cost, reliability, ease of
use, ingredient compatibility, ‘label friendliness’ and legal
status (Krog & Sparso, 2004; Stauffer, 1999). For these
reasons, there is no single emulsifier that is ideal for use in
every type of food product. Instead, the selection of the most
appropriate emulsifier or combination of emulsifiers for a
particular food product depends on the type and
concentration of other ingredients that it contains, the way
that it was produced, and the environmental conditions that it
experiences during its manufacture, storage and utilization.
There are limitations to the functional properties that
can be achieved using existing food emulsifiers
and the conventional method of creating emulsions, which
210 T. Aoki et al. / Food Hydrocolloids 19 (2005) 209–220
has led to research being carried out to find alternative
methods of improving emulsion stability by developing
novel emulsifier-based strategies. One strategy has been to
create covalent protein – polysaccharide complexes that
have good surface activity and provide improved protection
against environmental stresses (Dickinson, 2003; Schmitt,
Sanchez, Desobry-Banon, & Hardy, 1998). An alternative
strategy is to create an interfacial membrane around oil
droplets that consists of multiple layers of emulsifiers
and/or biopolymers using a layer-by-layer deposition
technique (Calvo, Remunan-Lopez, Vila-Jato, & Alonso,
1997; Caruso, 2001; Caruso & Schuler, 2000; Dickinson
& James, 2000; Faldt, Bergenstahl, & Claesson 1993;
Magdassi, Bach, & Mumcuoglu 1997; Moreau, Kim,
Decker, & McClements 2003; Ogawa, Decker, &
McClements 2003a,b; Shi & Caruso, 2001). In this
approach, an ionic emulsifier that rapidly adsorbs to the
surface of lipid droplets during homogenization is used to
produce a primary emulsion containing small droplets, then
an oppositely charged biopolymer is added to the system
that adsorbs to the droplet surfaces and produces
secondary emulsions containing droplets coated with a
two-layer interfacial membrane. This latter procedure can
be repeated to form oil droplets coated by interfacial
membranes containing three or more layers (Caruso, 2001;
Ogawa, Decker, & McClements 2004). Under certain
circumstances, emulsions containing oil droplets
surrounded by multilayered interfacial membranes have
been found to have better stability to environmental
stresses than conventional oil-in-water emulsions with
single-layered interfacial membranes.
The layer-by-layer deposition method therefore offers a
promising way to improve emulsion stability, but the
choice of an appropriate combination of emulsifier and
biopolymers is essential. In this study we examine the use
of an anionic surfactant (SDS), a cationic biopolymer
(chitosan), and an anionic biopolymer (pectin) to form the
interfacial membranes. SDS was chosen to represent
small-molecule anionic surfactants commonly used in
foods, such as fatty acid salts, lecithin, SLS, DATEM,
and CITREM (Krog & Sparso, 2004). Chitosan (cationic)
and pectin (anionic) were chosen because they are
examples of food-grade polysaccharides that have opposite
electrical charges. Chitosan is positively charged and
water-soluble at low pH (pKa < 6.5), but loses its charge
at higher pH and may precipitate from solution (Shahidi,
Arachchi, & Jeon, 1999; Skjak-Braek, Anthonsen, &
Sandford, 1989). Pectin is negatively charged at relatively
high pH values (pKa < 4 – 5), and is water-soluble
(Williams & Phillips, 2003). Previous studies have shown
that secondary emulsions stabilized by lecithin– chitosan
membranes have better stability than primary emulsions
stabilized by lecithin membranes to droplet aggregation
brought about by thermal processing (30 – 90 8C for
30 min), freeze – thaw cycling (2 10 8C for 22 h/30 8C
for 2 h) and exposure to high ionic strengths (, 500 mM
NaCl or CaCl2), as well as exhibiting less lipid oxidation
(peroxide formation) (Ogawa et al., 2003a,b).
Nevertheless, the secondary emulsions were only stable
at relatively low pH values (, pH 5) and required
cryoprotectants to inhibit freeze –thaw instability, which
would limit their application in many industrial products.
The objective of the present investigation was to determine
whether we could improve the freeze – thaw and pH
stability of lecithin– chitosan emulsions by incorporating
pectin as a third layer.
2. Experimental
2.1. Materials
Sodium dodecyl sulfate (SDS), chitosan (from crab
shells, minimum 85% deacetylated), analytical grade
sodium chloride, hydrochloric acid, sodium hydroxide,
sodium azide, acetic acid and sodium acetate were obtained
from the Sigma Chemical Company (St Louis, MO).
Pectin (extracted from citrus) was purchased from Sigma-
Aldrich Co. (St Louis, MO). As stated by the manufacturer,
the degree of esterification of the pectin was 59%. Corn oil
was purchased from a local supermarket and used without
further purification. Distilled and de-ionized water was used
for the preparation of all solutions.
2.2. Solution preparation
An emulsifier solution was prepared by dispersing
20 mM SDS in acetate buffer (100 mM acetic acid, adjusted
to pH 3.0 with 1 M HCl). Chitosan and pectin solutions were
prepared by dispersing 0.2 wt% chitosan and 0.4 wt% pectin
in acetate buffer. Sodium azide stock of 0.2 wt% was
prepared in acetate buffer. The pH of these solutions was
adjusted back to 3.0 using 1 M HCl, and then the solutions
were stirred for about 1 day to ensure complete dissolution
of these materials.
2.3. Primary emulsion preparation
A preliminary experiment was carried out to determine
the optimum SDS concentration required to create the
primary emulsions, i.e. the minimum amount of SDS
required to produce emulsions that were stable to droplet
aggregation. Primary emulsions were prepared by
homogenizing 5 wt% corn oil with 95 wt% aqueous
emulsifier solution (1 –10 mM SDS) in a high-speed blender
(M133/1281-0, Biospec Products, Inc., ESGC, Switzerland)
followed by sonication for 30 s at a frequency of 20 kHz,
amplitude of 40% and duty cycle of 0.5 s (Model 500,
Sonic Disembrator, Fisher Scientific, Pittsburgh, PA).
The primary emulsions were then stored at room
temperature for 24 h before being analyzed to determine
the free SDS concentration and their stability to droplet
 T. Aoki et al. / Food Hydrocolloids 19 (2005) 209–220
aggregation. The optimum surfactant concentration was
established to be 5 mM SDS (see Section 3.1), which was
used to prepare the primary emulsions in all subsequent
experiments.
2.4. Secondary emulsion preparation
A primary emulsion was prepared by homogenizing
5 wt% corn oil with 95 wt% aqueous emulsifier solution
(5 mM SDS) in a high-speed blender (M133/1281-0,
Biospec Products, Inc., ESGC, Switzerland) followed by
three passes at 5000 psi through a two-stage high-pressure
valve homogenizer (LAB 1000, APV-Gaulin, Wilmington,
MA). This emulsion was diluted with aqueous chitosan
solutions to form secondary emulsions with varying
compositions: 1 wt% corn oil, 1 mM SDS, 100 mM acetic
acid, and 0– 0.040 wt% chitosan (pH 3.0). The secondary
emulsions were stored at room temperature for 24 h before
being analyzed.
2.5. Tertiary emulsion preparation
A secondary emulsion was prepared as described in
Section 2.4, that had a final concentration of 1 wt% corn oil,
1 mM SDS, 0.024 wt% chitosan, 100 mM acetic acid
(pH 3.0). This system was stirred for 1 h using a magnetic
stirrer at ambient temperature to thoroughly mix the primary
emulsion with the chitosan. As observed in previous studies,
some droplet flocculation was observed in the secondary
emulsion prepared using this method, so the emulsions was
passed three times through a high-pressure value
homogenizer at a pressure of 5000 psi to disrupt these
flocs (Ogawa et al 2003a,b). Tertiary emulsions were
formed by diluting the secondary emulsion with aqueous
pectin solution to produce a series of emulsions with
different pectin concentrations: 0.2 wt% corn oil, 0.2 mM
SDS, 0.0048 wt% chitosan, 100 mM acetic acid and
0 –0.040 wt% pectin (pH 3.0). The tertiary emulsions were
stored at room temperature for 24 h before being analyzed.
2.6. Free SDS measurements
A surfactant selective electrode (SSE) was used to
measure electro-motive force (EMF) of free SDS. The SSE
consisted of a surfactant selective electrode, a double
junction reference electrode, and a pH meter (Thermo Orion,
Beverly, MA). The EMF of primary emulsions prepared
using different initial surfactant concentrations was
measured by dipping the SSE electrode directly into the
emulsions (1 –10 mM SDS). The free SDS concentration in
the emulsions was then calculated from a standard curve
prepared using known concentrations of SDS dissolved in
acetate buffer (100 mM, pH 3.0).
211
2.7. Particle size measurements
Emulsions were diluted to a droplet concentration of
approximately 0.005 wt% oil using acetate buffer solution
to avoid multiple scattering effects. The particle size
distribution of the emulsions was measured using a laser
light scattering instrument (Mastersizer, Malvern
Instruments Ltd., Worcs., UK). This instrument measures
the angular dependence of the intensity of laser light
(l ¼ 632:8 nm) scattered by a dilute emulsion, and then
finds the particle size distribution that gives the best
agreement between theoretical predictions and experimental
measurements. A refractive index ratio of 1.08 was used in
the calculations of the particle size distribution. It should
be noted that the theory used to calculate the particle size
distribution assumes that the particles are spherical and
homogeneous, and therefore the data obtained on emulsions
that contained flocs should be treated with caution because
they are non-spherical and non-homogenous. Particle size
measurements are reported as the volume-surface mean
diameter: d32 ¼ Sn3i =Sn2i ; where ni is the number of droplets
of diameter di : Mean particle diameters were calculated as
the average of measurements made on at least three
samples, with standard deviations being less than 10%.
2.8. z-Potential measurements
Emulsions were diluted to a droplet concentration of
approximately 0.005 wt% oil using buffer solution to avoid
multiple scattering effects. Diluted emulsions were
injected directly into the measurement chamber of a
particle electrophoresis instrument (ZEM5003, Zetamaster,
Malvern Instruments, Worcs., UK). The z-potential
was then determined by measuring the direction and
velocity of droplet movement in a well-defined electric
field. The z-potential measurements are reported as the
mean and standard deviation of two separate injections, with
five readings made per injection.
2.9. Creaming stability measurements
Approximately 3.5 g samples of diluted emulsion
(0.005 wt% oil) were transferred into 1-cm path length
plastic spectrophotometer cuvettes, and then stored at room
temperature for 1 day. The turbidity (at 600 nm) of the
emulsions was then measured using an UV – visible
spectrophotometer. The light beam passed through the
emulsions at a height that was 10 mm from the cuvette
bottom, i.e. about 30% of the emulsion’s height. The oil
droplets in the emulsions moved upwards due to
gravity, which led to the formation of a relatively clear
droplet-depleted serum layer at the bottom of the cuvette.
An appreciable decrease in emulsion turbidity was therefore
an indication of the fact that the serum layer had risen to at
least 30% of the emulsion’s height.
212 T. Aoki et al. / Food Hydrocolloids 19 (2005) 209–220

2.10. Emulsion environmental stresses

We compared the influence of various kinds of

environmental stresses on the z-potential, mean particle
diameter and creaming stability of primary, secondary and
tertiary emulsions.
Thermal processing. The primary and secondary emul-
sions were diluted with acetic acid buffer (pH 3.0) to adjust
the oil concentration to the same value as in the tertiary
emulsion (0.2 wt%). Emulsion samples (10 ml) were
transferred into glass test tubes, which were then incubated
in a water bath for 30 min at different temperatures ranging
from 30 to 90 8C. After incubation the emulsion samples
were immediately placed at room temperature, where they
were stored prior to analysis.
Freeze – thaw cycling stability. The primary and Fig. 1. Dependence of free SDS concentration on total SDS concentration in
primary emulsions (5 wt% corn oil, 100 mM acetic acid buffer, pH 3.0)
secondary emulsions were diluted with acetic acid buffer measured using a surfactant selective electrode.
(pH 3.0) to adjust the oil concentration to the same value as
in the tertiary emulsion (0.2 wt%). These emulsion samples
emulsion was studied by measuring the EMF with a
(10 ml) were transferred into cryogenic test tubes and were
surfactant selective electrode (SSE). A standard curve of
incubated in a 2 20 8C freezer for 22 h. After incubation the
EMF versus free SDS was obtained by measuring the EMF
emulsion samples were thawed by incubating them in a
of a series of solutions with different SDS concentrations
water bath at 30 8C for 2 h. This freeze –thaw cycle was
(100 mM acetic acid buffer, pH 3.0). The free SDS
repeated six times and its influence on emulsion properties
concentration in primary emulsions (5 wt% corn oil,
was measured after each cycle.
100 mM acetic acid buffer, pH 3.0) containing different
NaCl stability. Primary, secondary and tertiary emulsion
total SDS concentrations (1 – 10 mM) were then determined
samples were diluted with acetic acid buffer (pH 3.0) to
from EMF measurements using the standard curve (Fig. 1).
obtain the same final oil concentration (0.05 wt%),
Fig. 1 shows that around 2– 3 mM of SDS was adsorbed to
but different NaCl concentrations (0 – 500 mM).
the surfaces of the emulsion droplets, and above this value
The influence of NaCl concentration (0 – 500 mM) on the
the rest of the SDS was present in the continuous phase.
z-potential, mean particle diameter, and creaming stability
Ideally, we wanted to minimize the concentration of non-
of diluted primary, secondary and tertiary emulsions at pH
adsorbed SDS in the primary emulsions (to avoid its
3.0 was measured after 24 h.
interaction with chitosan), but we wanted to use sufficient
pH stability. Primary, secondary and tertiary emulsion
SDS to ensure that small droplets were formed that were
samples were diluted with acetate buffer (pH 3.0 –8.0) to
obtain the same final oil concentration (0.05 wt%), relatively stable to aggregation. We found that if the total
but different pH values. The influence of pH (3.0 – 8.0) on SDS concentration was too low (, 4 mM), then the droplets
the z-potential, mean particle diameter, and creaming formed were relatively large or were unstable to
stability of diluted primary, secondary and tertiary coalescence (i.e. oiling off was observed on the surface of
emulsions was measured after 1 day and 10 days storage. the emulsions). We therefore decided to use 5 mM SDS to
prepare the primary emulsions used in the subsequent
experiments.
3. Results and discussion
3.2. Influence of chitosan concentration on droplet
3.1. Influence of SDS concentration on droplet characteristics of secondary emulsions
characteristics of primary emulsions
The purpose of these experiments was to establish the
The purpose of these experiments was to determine the optimum chitosan concentration required to prepare stable
optimum SDS concentration required to prepare the primary secondary emulsions. The z-potential, mean particle
emulsions. If the SDS concentration is too low, it would not diameter and turbidity of secondary emulsions (1 wt%
be possible to form stable emulsions, but if the SDS corn oil, 1 mM SDS, 100 mM acetic acid buffer, pH 3.0)
concentration was too high there would be a lot of containing different chitosan concentrations (0 –0.040 wt%)
non-adsorbed surfactant present in the aqueous phase that were measured after 24 h storage at room temperature
could interact with the biopolymers subsequently used to (Fig. 2). In the absence of chitosan, the net charge on the
create the multiple-layered interfacial membranes. emulsion droplets was , 2 60 mV, because the SDS used
The adsorption of SDS to oil droplets in the primary to stabilize the droplets in the primary emulsion has
 T. Aoki et al. / Food Hydrocolloids 19 (2005) 209–220 213

Fig. 2. (a) Dependence of particle electrical charge (z-potential) on chitosan concentration for secondary emulsions (1 wt% corn oil, 1 mM SDS, 100 mM
acetic acid, pH 3.0). (b) Dependence of mean particle diameter ðd32 Þ on chitosan concentration for secondary emulsions (1 wt% corn oil, 1 mM SDS, 100 mM
acetic acid, pH 3.0). The photographs at the top show micrographs of selected emulsions with different chitosan concentrations obtained by optical
microscopy. (c) Dependence of emulsion creaming stability on chitosan concentration for secondary emulsions (1 wt% corn oil, 1 mM SDS, 100 mM acetic
acid, pH 3.0). The creaming stability was determined as turbidity (at 600 nm) measured at 30% of emulsion height after 24 h storage: creaming instability is
indicated by a low turbidity. The photograph shown above highlights the instability of the emulsions to creaming at intermediate chitosan concentrations
(taken after sonication).
214 T. Aoki et al. / Food Hydrocolloids 19 (2005) 209–220
a negative charge at pH 3 (Fig. 2a). The net charge on the
droplets switched from negative to positive when the
chitosan concentration in the emulsions was increased,
and eventually reached a relatively constant positive value
(, þ 60 mV) when the chitosan concentration exceeded
about 0.020 wt% (Fig. 2a), presumably because the droplets
become saturated with chitosan. This change in z-potential
suggests that cationic chitosan molecules adsorbed to the
surface of anionic SDS-coated emulsion droplets.
In the absence of chitosan, the mean particle diameter of
the emulsion droplets was , 0.3 mm and no creaming was
observed at 24 h storage, which indicated that SDS was
effective at generating small emulsion droplets during
homogenization. At chitosan concentrations . 0.012 wt%
there was a large increase in mean particle diameter and a
decrease in turbidity, which was attributed to extensive
droplet aggregation and creaming (Fig. 2b and c).
The maximum amount of droplet aggregation occurred at
a chitosan concentration of 0.016 wt%. In fact, droplet
aggregation was so extensive around this chitosan
concentration that we were unable to make reliable particle
size or z-potential measurements, either because the
aggregates were too large or because their concentration
was too small to give a scattering pattern that was
sufficiently different from that of the background. Individual
particles within these emulsions could be distinctly
observed by eye, which suggested that they were at least
100 mm in diameter. One might have expected that at
sufficiently high chitosan concentrations the emulsions
would have been stable to droplet aggregation because of
the large electrostatic repulsion between the highly
positively charged droplets. It is likely that the droplets
were held together by chitosan molecules that were
adsorbed to the surface of more than one droplet (Pinotti,
Bevilacqua, & Zaritzky 1997, 1999, 2001; Pinotti &
Zaritzky, 2001). This is not surprising, since charged
polymers are known to induce bridging flocculation of
oppositely charged colloidal particles (Dickinson, 1992,
2003; Friberg et al., 2004; Von Goeler & Muthukumar,
1994). During the initial stages of mixing of the chitosan
solution with the primary emulsion, there would have been
many droplets present with completely negative surfaces;
hence, it is likely that chitosan molecules could adsorb to the
surface of two or more of these droplets simultaneously.
A similar phenomenon has been observed in previous
studies (Von Goeler & Muthukumar, 1994).
In previous studies we found that sonication could be
used to effectively breakdown flocs in secondary emulsions
(Ogawa et al., 2003a,b). We therefore examined the
possibility of using the same method for producing
secondary emulsions with improved stability to droplet
flocculation in this study. Emulsion samples (, 10 g) were
sonicated for 30 s at a frequency of 20 kHz, an amplitude of
40%, and a duty cycle of 0.5 s (Model 500, Sonic
Dismembrator, Fisher Scientific, Pittsburgh, PA), and then
the z-potential mean, mean particle diameter and turbidity
of the emulsions were measured (Fig. 2). Sonication caused
a pronounced decrease in the mean particle diameter of all
the emulsions (Fig. 2b), which suggests that it was
capable of generating forces sufficiently large to either
physically cleave chitosan molecules or to cause them to
become detached from all but one of the droplets.
Despite the application of sonication, emulsions containing
0.012 – 0.016 wt% chitosan still exhibited extensive
aggregation (Fig. 2b and c), presumably because the
electrostatic repulsion between the droplets was insufficient
to prevent flocculation. At relatively high chitosan
concentrations (. 0.02 wt%), where the droplets had
relatively high positive charges (, þ 60 mV), the
measured mean particle diameters were similar to those of
the primary emulsion (, 0.3 mm). In these systems, the
electrical charge on the droplets must have been large
enough to prevent them from coming back into close
contact after the flocs had been disrupted by sonication.
These experiments showed that emulsions with non-aggre-
gated droplets could be produced by applying sonication to
secondary emulsions containing sufficiently high levels of
chitosan. A chitosan concentration of 0.024 wt% was
therefore chosen to produce the secondary emulsions used
in the remaining experiments.
3.3. Influence of pectin concentration on droplet
characteristics of tertiary emulsions
The purpose of these experiments was to determine the
optimum amount of pectin required to produce stable
tertiary emulsions. The z-potential, particle diameter and
turbidity of tertiary emulsions (0.2 wt% corn oil, 0.2 mM
SDS, 0.0012 wt% chitosan, 100 mM acetic acid buffer,
pH 3.0) containing different pectin concentrations
(0 –0.04 wt%) were measured 24 h after preparation (Fig. 3).
In the absence of pectin, the net charge on the emulsion
droplets was , þ 60 mV due to the presence of cationic
chitosan on the droplet surfaces. The net charge on the
droplets changed from positive to negative as the pectin
concentration in the emulsions was increased (Fig. 3a).
Charge neutralization occurred when the pectin
concentration was , 0.010 wt%. The negative charge on
the droplets reached a constant value when the pectin
concentration exceeded , 0.020 wt%. These measurements
indicated that negatively charged pectin adsorbed to the
surface of the positively charged SDS – chitosan stabilized
emulsion droplets. The negatively charged pectin molecules
continued to adsorb to the surfaces of the emulsion droplets,
even though the droplets had no net charge or were
negatively charged, up to a certain saturation level (Fig. 3a).
The ability of charged polyelectrolyte to adsorb to the
surface of oppositely charged colloidal particles and cause
charge reversal is well established in the literature
(Chodanowski & Stoll, 2001; Dobrynin, 2001; Jonsson &
Linse, 2001; Kong & Muthukumar, 1998; Netz & Joanny,
1999; Von Goeler & Muthukumar, 1994).
 T. Aoki et al. / Food Hydrocolloids 19 (2005) 209–220 215

Fig. 3. (a) Dependence of particle electrical charge (z-potential) on pectin concentration for tertiary emulsions (0.2 wt% corn oil, 0.2 mM SDS, 0.0012 wt%
chitosan, 100 mM acetic acid buffer, pH 3.0). (b). Dependence of mean particle diameter ðd32 Þ on pectin concentration for tertiary emulsions (0.2 wt% corn oil,
0.2 mM SDS, 0.0012 wt% chitosan, 100 mM acetic acid buffer, pH 3.0). (c) Dependence of emulsion creaming stability on pectin concentration for tertiary
emulsions (0.2 wt% corn oil, 0.2 mM SDS, 0.0012 wt% chitosan, 100 mM acetic acid buffer, pH 3.0). The creaming stability was determined as turbidity
(at 600 nm) measured at 30% of emulsion height after 24 h storage: creaming instability is indicated by a low turbidity. The photograph shown above highlights
the instability of the emulsions to creaming at intermediate pectin concentrations (taken after sonication).

There was a significant increase (from d32 < 0:3 to
1.6 mm) in the mean particle diameter and decrease in the
turbidity (from . 0.7 to , 0.1 cm21) of the tertiary emulsions
when the pectin concentration was increased from 0 to
0.006 wt% (Fig. 3b and c), which corresponded to the region
where charge neutralization occurred (Fig. 3a). Nevertheless
at higher pectin concentration (0.02 – 0.04 wt%)
the emulsions contained highly negatively charged particles
and were stable to droplet aggregation, even in the absence of
sonication. In the subsequent experiments we therefore used
a pectin concentration of 0.04 wt% to prepare the tertiary
emulsions.
3.4. Influence of thermal processing on properties
of primary, secondary and tertiary emulsions
The purpose of these experiments was to examine the
influence of thermal processing on the stability of SDS
(primary emulsion), SDS – chitosan (secondary emulsion)
and SDS – chitosan – pectin (tertiary emulsion) coated
droplets. Primary, secondary and tertiary emulsions with
the same oil concentration (0.2 wt%) were held at
temperatures ranging from 30 to 90 8C, cooled to room
temperature, and then stored for 24 h. The influence of
thermal processing on the mean particle diameter and
creaming stability of primary, secondary and tertiary
emulsions was measured after thermal processing.
There was no significant change in the mean particle
diameter (d32 ¼ 0:27 ^ 0.03 mm) and turbidity (p # 5%)
of the primary (t ¼ 20:06 ^ 0.01 cm 21), secondary
(t ¼ 0:12 ^ 0.01 cm21) and tertiary emulsions (t ¼ 0:16 ^
0.01 cm21) upon heat treatment, which indicated that they
were all stable to thermal processing in the temperature
range used in this study.
3.5. Influence of freeze –thaw cycling on properties
of primary, secondary and tertiary emulsions
The purpose of these experiments was to examine the
freeze –thaw stability of primary, secondary and tertiary
emulsions. Emulsions were frozen at 2 20 8C for 22 h, then
thawed at 30 8C for 2 h in a water bath. The influence of
freeze –thaw cycling (up to 6 times) on the mean particle
diameter and creaming stability of diluted primary,
secondary and tertiary emulsions at pH 3.0 was measured
(Fig. 4). The primary and secondary emulsions were highly
unstable to freeze – thaw cycling, there being an appreciable
increase in mean particle diameter (Fig. 4a) and rapid
216 T. Aoki et al. / Food Hydrocolloids 19 (2005) 209–220

Fig. 4. (a) Dependence of mean particle diameter of primary, secondary and tertiary emulsions on number of freeze–thaw cycles. (b) Dependence of emulsion
creaming stability of primary, secondary and tertiary emulsions on number of freeze– thaw cycles. The creaming stability was determined as turbidity
(at 600 nm) measured at 30% of emulsion height after 24 h storage: creaming instability is indicated by a low turbidity. A photograph of the primary, secondary
and tertiary emulsion after 1 freeze–thaw cycle is shown above.

creaming (Fig. 4b) after the first cycle, suggesting extensive
droplet aggregation. Nevertheless, the particle size data on
the primary and secondary emulsions after freezing and
thawing should be treated with caution because they were
either highly coalesced (exhibiting oiling off) or highly
flocculated, respectively, thereby making reliable light
scattering measurements difficult to make. On the other
hand, the mean particle diameter of the particles in the
tertiary emulsions remained stable after 6 freeze – thaw
cycles (, 0.3 ^ 0.05 mm), and there was no evidence of
creaming (the turbidity remained fairly constant).
A number of physicochemical phenomena that occur
during frozen storage may account for the observed influence
of freeze –thaw cycling on emulsion stability. First, water
crystallization occurred when the emulsions were placed in
the freezer. As more and more water crystallized the droplets
would have been forced closer together (Saito et al., 1999)
and there may not have been sufficient free water present to
fully hydrate the droplet surfaces (Ausborn et al., 1994;
Komatsu, Okada, & Handa 1997; Strauss & Hauser, 1986),
thus favoring droplet – droplet interactions. Second, ice
crystallization leads to an increase in the ionic strength of
any freeze-concentrated non-frozen aqueous phase
surrounding the emulsion droplets (Komatsu et al., 1997),
which may have promoted droplet –droplet interactions.
Third, it is possible that ice crystals formed during freezing
 T. Aoki et al. / Food Hydrocolloids 19 (2005) 209–220 217

may have penetrated into the oil droplets and disrupted their
interfacial membranes, thus making them more prone to
coalescence. Fourth, cooling may have caused some of the fat
in the emulsion droplets to crystallize, which may have
promoted partial coalescence due to penetration of a fat
crystal from one droplet through the membrane of another
droplet (Harada & Yokomizo, 2000; Vanapalli, Palanuwech,
& Coupland 2002).
There are a number of possible reasons that the tertiary
emulsions were more stable to freeze –thaw cycling than
the primary and secondary emulsions. First, the interfacial
layer in the tertiary emulsions is thicker than that in the
primary and the secondary emulsions, so there will be a
greater short-range steric repulsion between the droplets
that prevents them from coming close enough to
coalesce (Hunter, 1986; Israelachvili, 1992). Second, it
may be more difficult for fat or ice crystals to rupture the
thicker SDS – chitosan– pectin membranes than the thinner
SDS or SDS – chitosan membranes, thereby making the
droplets in the tertiary emulsion more stable to
droplet coalescence or partial coalescence (Goff, 2000;
McClements, 2001). Third, droplets coated with SDS –chi-
tosan –pectin membranes are more stable to aggregation at
high salt concentrations than droplets coated with SDS or
SDS –chitosan membranes, which may have been important
if the ionic strength of the freeze-concentrated solution
surrounding the droplets increased (see Section 3.6).
3.6. Influence of ionic strength on properties of primary,
secondary and tertiary emulsions
The purpose of these experiments was to examine the
influence of ionic strength on the stability of primary,
secondary and tertiary emulsions. The influence of NaCl
concentration (0 – 500 mM) on the z-potential, mean particle
diameter and turbidity of diluted primary, secondary and
tertiary emulsions at pH 3.0 was measured (Fig. 5).
The z-potential of the SDS-stabilized droplets in the primary
emulsions was negative at all ionic strengths, but the
magnitude of the z-potential decreased as the NaCl
concentration was increased which can be attributed to
electrostatic screening (Hunter, 1986). The z-potential of
the chitosan –SDS stabilized droplets in the secondary
emulsions was positive at all ionic strengths but the
magnitude of the z-potential decreased as the NaCl
concentration was increased, which can also be attributed

Fig. 5. (a) Dependence of particle electrical charge (z-potential) on NaCl concentration for primary, secondary and tertiary emulsions at pH 3. (b) Dependence
of mean particle diameter ðd32 Þ on NaCl concentration for primary, secondary and tertiary emulsions at pH 3. (c) Dependence of emulsion creaming stability on
NaCl concentration for primary, secondary and tertiary emulsions at pH 3. The creaming stability was determined as turbidity (at 600 nm) measured at 30% of
emulsion height after 24 h storage: creaming instability is indicated by a low turbidity. The photograph shown above highlights the instability of the primary
emulsions to creaming at high NaCl concentrations.
218 T. Aoki et al. / Food Hydrocolloids 19 (2005) 209–220

to electrostatic screening effects (Hunter, 1986). It is also

possible that some of the decrease of charge on the emulsion
droplets in the secondary emulsions was due to desorption
of chitosan from the emulsion droplet surfaces when the
ionic strength increased. This could have occurred because
the electrostatic attraction between the negatively charged
SDS molecules and the positively charged chitosan
molecules was weakened at higher ionic strengths,
thus allowing desorption of some of the cationic
biopolymer. The z-potential of the pectin – chitosan– SDS
stabilized droplets in the tertiary emulsion was negative at
all ionic strengths but the magnitude of the z-potential did
not change appreciatively as the NaCl concentration was
increased (Fig. 5a), which suggested that pectin did not
desorb from the emulsion droplet surfaces at higher ionic
strength. In addition, one would have expected a decrease in
z-potential due to electrostatic screening effects, which was
not observed. It is possible that the thickness of this
adsorbed interfacial layer changed with ionic
strength, which kept the magnitude of the surface potential
relatively constant.
The secondary and tertiary emulsions were relatively
stable to droplet aggregation at all ionic strengths
(0 – 500 mM NaCl), as demonstrated by the relative
independence of turbidity on NaCl concentration (Fig. 5c).
On the other hand the primary emulsion became strongly
aggregated at high ionic strength (500 mM NaCl), as shown
by the large decrease in turbidity (Fig. 5c). In the case of the
primary emulsion, droplet aggregation was probably caused
by the reduction of repulsive interactions between the
droplets due to electrostatic screening, but it may also have
been due to the ability of salt to change the optimum
curvature of the SDS (Hunter, 1986; Israelachvili, 1992).
The secondary and tertiary emulsions were probably
stable to droplets aggregation at high salt because the
relatively thick interfacial membranes provided good steric
stabilization.

3.7. Influence of pH on stability of primary, secondary
and tertiary emulsions
The purpose of these experiments was to examine the
influence of pH on the stability of primary, secondary and
tertiary emulsions. The influence of pH on the z-potential,
mean particle diameter, and turbidity of diluted primary,
secondary and tertiary emulsions at pH 3.0 – 8.0 was
measured after they had been stored at room temperature
for 1 day and 10 days after preparation (Fig. 6).
The z-potential of the SDS stabilized droplets in primary
emulsions was negative at all pH values, but the magnitude
of the z-potential decreased when the pH was increased
from 3 to 8, probably due to electrostatic screening effects
associated with addition of the alkali used to adjust pH
(Hunter, 1986). The z-potential of the chitosan – SDS
stabilized droplets in the secondary emulsions was positive
at low pH (, pH 6), but became negative at higher values
Fig. 6. (a) Dependence of particle electrical charge (z-potential) on pH
concentration for primary, secondary and tertiary emulsions. (b) Depen-
dence of mean particle diameter ðd32 Þ on pH for primary, secondary and
tertiary emulsions. (c) Dependence of emulsion creaming stability on pH
for primary, secondary and tertiary emulsions after 10 days storage. The
creaming stability was determined as turbidity (at 600 nm) measured at
30% of emulsion height after 24 h storage: creaming instability is indicated
by a low turbidity.
(pH 7 and 8). The reversal of charge on the emulsion
droplets suggests that the chitosan was desorbed from the
emulsion droplet surfaces when the pH was increased above
6. The cationic groups (– NHþ3 ) on chitosan typically have a
pKa value around pH 6.3 –7; hence, the chitosan loses its
charge around this pH (Ogawa et al., 2004). The z-potential
of the pectin – chitosan– SDS stabilized droplets in the
tertiary emulsion was negative at all pH, which suggested
that the chitosan layer did not desorb from the surface of
the emulsion droplets at higher pH values even though it
 T. Aoki et al. / Food Hydrocolloids 19 (2005) 209–220
did in the secondary emulsions. This may have been because
the pKa value of the positively charged groups on the
chitosan molecules was increased appreciably when the
chitosan was sandwiched between two negatively charged
biopolymer layers, as has been reported for other
polyelectrolytes (Burke & Barrett, 2003a,b). The increase
in the magnitude of the negative charge with increasing pH,
was probably because of the increase in the negative charge
on the pectin molecules (pKa , 4.5) and the decrease in
the positive charge on the chitosan molecules (pKa , 6.5) as
the pH increased.
The droplets in the primary, secondary and tertiary
emulsions were stable to droplet aggregation at pH 3 –5
when stored for 10 days. But aggregation occurred in
primary and secondary emulsion after 10 days storage at pH
6 –8 as demonstrated by the rapid decrease in creaming
stability in these emulsions (Fig. 6b). This was probably
because the decrease in droplet charge (Fig. 6a) reduced the
electrostatic repulsion between the droplets, which led to
extensive droplet flocculation. The tertiary emulsion was
stable to creaming and droplet aggregation over the whole
pH range because of the relatively strong electrostatic and
steric repulsion associated with the relatively thick and
electrically charged three-layer interfacial membrane.
In addition, the creaming stability may have been improved
because the multiple layers increased the overall density of
the droplets, thereby reducing the density contrast between
the dispersed and continuous phases and decreasing the
driving force for gravitational separation (Friberg et al.,
2004; McClements, 1999; Stauffer, 1999).
4. Conclusions
This study has shown that stable emulsions containing
tri-layered lipid droplets can be prepared using a simple and
cost-effective method. Initially, a primary emulsion
containing small droplets was produced by homogenization
of oil, water, and an anionic emulsifier (SDS at pH 3.0).
A secondary emulsion containing cationic lipid droplets
coated with a SDS –chitosan membrane was then produced
by mixing a cationic polysaccharide (chitosan at pH 3) with
the primary emulsion and agitating to disrupt any flocs
formed. A tertiary emulsion containing anionic droplets
coated with a SDS – chitosan – pectin membrane was then
produced by mixing an anionic polysaccharide (pectin at pH
3) with the secondary emulsion, without applying mechan-
ical agitation.
This study also compared the stability of SDS-coated
(primary), SDS – chitosan-coated (secondary) and
SDS – chitosan – pectin-coated (tertiary) oil droplets to
droplet aggregation induced by thermal processing,
freeze – thaw cycling, NaCl and pH. Our results clearly
show that emulsion stability could be dramatically
improved by coating the droplets with two or more layers.
The droplets in the tertiary emulsions had good stability to
219
droplet aggregation and creaming at high temperatures
(90 8C), several freeze – thaw cycles, high salt
concentrations (NaCl 500 mM), and a wide range of pH
values (pH 3 –8). The improved stability of the tertiary
emulsions can be attributed to the ability of multilayered
interfacial membranes to either increase the repulsive
colloidal interactions between the droplets (e.g. electrostatic
and steric) and/or to increase the resistance of the membrane
to rupture. The tertiary emulsions may have a number of
potential applications in the food industry for specialized
applications. For example, the thick interfacial membranes
may be useful in protecting lipid components, for controlled
flavor release or for improving the stability of emulsions to
environmental stresses, such as freezing and heating.
Finally, we should point out some of the current
limitations of the layer-by-layer deposition method for
application in the food industry. The final emulsions used in
this study had relatively low droplet concentrations
(0.2 wt%), which may be suitable for certain applications
in the food industry (e.g. beverages), but not for others
(e.g. dressings, sauces, soups). One of the major problems
with preparing more concentrated emulsions is the
tendency for the droplets to flocculate due to a bridging
mechanism. We are currently investigating methods of
optimizing the preparation procedures so that more
concentrated emulsions can be prepared. Another potential
limitation of this technology is the number of additional
steps required to prepare the multilayered emulsions
compared to conventional emulsions, which will increase
production costs and therefore limit its application to certain
specialized products.
Acknowledgements
This material is based upon work supported by the
National Research Initiative, Competitive Grants Program,
United States Department of Agriculture, and the CREES,
IFAFS Program, United States Department of Agriculture
(Award Number 2002-35503-12296). We also thank the
Tokyo University of Marine Science and Technology
(Japan) for financial support of Tomoko Aoki.
References
Ausborn, M., Schreier, H., Brezesinski, G., Fabian, H., Meyer, H. W., &
Nuhn, P. (1994). The protective effect of free and membrane-bound
cryoprotectants during freezing and freeze-drying of liposomes.
Journal of Controlled Release, 30, 105 –116.
Burke, S. E., & Barrett, C. J. (2003a). pH-responsive properties of
multilayered poly(L -lysine)/hyaluronic acid surfaces. Biomacromole-
cules, 4, 1773–1783.
Burke, S. E., & Barrett, C. J. (2003b). Acid–base equilibria of weak
polyelectrolytes in multilayer thin films. Langmuir, 19, 3297–3303.
Calvo, P., Remunan-Lopez, C., Vila-Jato, J. L., & Alonso, M. J. (1997).
Development of positively charged colloidal drug carriers:
220 T. Aoki et al. / Food Hydrocolloids 19 (2005) 209–220
Chitosan-copolyester nanocapsules and submiron-emulsions.
Colloid and Polymer Science, 275, 46–53.
Caruso, F. (2001). Generation of complex colloids by polyelectrolyte-
assisted electrostatic self-assembly. Australian Journal of Chemistry,
54, 349 –353.
Caruso, F., & Schuler, C. (2000). Enzyme multilayers on colloid particles:
Assembly, stability, and enzymatic activity. Langmuir, 16, 9595–9603.
Charlambous, G., & Doxastakis, G. (1989). Food emulsifiers: Chemistry,
technology, functional properties and applications. Amsterdam:
Elsevier.
Chodanowski, P., & Stoll, S. (2001). Polyelectrolyte adsorption on charged
particles: Ionic concentration and particle size effects—A Monte Carlo
approach. Journal of Chemical Physics, 115, 4951–4960.
Dickinson, E. (1992). An introduction to food colloids. Oxford:
Oxford Science Publishers.
Dickinson, E. (2003). Hydrocolloids at interfaces and the influence on the
properties of dispersed systems. Food Hydrocolloids, 17(1), 25–39.
Dickinson, E., & James, J. D. (2000). Influence of high-pressure treatment
on b-lactoglobulin–pectin associations in emulsions and gels. Food
Hydrocolloids, 14, 365–376.
Dobrynin, A. V. (2001). Effect of solvent quality on polyelectrolyte
adsorption at an oppositely charged surface. Journal of Chemical
Physics, 115, 8145–8153.
Faldt, P., Bergenstahl, B., & Claesson, P. M. (1993). Stabilization by
chitosan of soybean oil emulsions coated with phospholipid and
glycholic acid. Colloids and Surfaces A, 71, 187–195.
Friberg, S., Larsson, K., & Sjoblom, J. (2004). Food emulsions (4th ed.).
New York: Marcel Dekker.
Garti, N., & Reichman, D. (1993). Hydrocolloids as food emulsifiers and
stabilizers. Food Microstructure, 12, 411 –426.
Goff, H. D. (2000). Controlling ice-cream structure by examining fat:
Protein interactions. Australian Journal of Dairy Technology, 55,
78–81.
Harada, T., & Yokomizo, K. (2000). Demulsification of oil-in-water
emulsion under freezing conditions: Effect of crystal structure
modifier. Journal of the American Oil Chemical Society, 77,
859– 863.
Hunter, R. J. (1986) (Vol. 1). Foundations of colloid science, Oxford:
Oxford University Press.
Israelachvili, J. N. (1992). Intermolecular and surface forces. London:
Academic Press.
Jonsson, M., & Linse, P. (2001). Polyelectrolyte –macroion complexation:
Effect of linear charge density and macroion charge. Journal of
Chemical Physics, 115, 3406–3418.
Komatsu, H., Okada, S., & Handa, T. (1997). Suppressive effects of salts
on droplet coalescence in a commercially available fat emulsion
during freezing for storage. Journal of Pharmaceutical Science, 86,
497– 502.
Kong, K. Y., & Muthukumar, M. (1998). Monte Carlo study of adsorption
of a polyelectrolyte onto charged spheres. Journal of Chemical Physics,
109, 1522–1527.
Krog, N. J., & Sparso, F. V. (2004). Food emulsifiers: Their chemical and
physical properties. In S. Friberg, K. Larsson, & J. Sjoblom (Eds.),
Food emulsions (4th ed.). New York: Marcel Dekker, chapter 2.
Magdassi, S., Bach, U., & Mumcuoglu, K. Y. (1997). Formation of
positively charged microcapsules based on chitosan–lecithin inter-
actions. Journal of Microencapsulation, 14, 189–195.
McClements, D. J. (1999). Food emulsions: Principles, practice and
techniques. Boca Raton, FL: CRC Press.
McClements, D. J. (2001). Estimation of steric exclusion and differential
interaction contributions to protein transfer free energies in aqueous
cosolvent solutions. Food Hydrocolloids, 15, 355 –363.
Moreau, L., Kim, H.-J., Decker, E. A., & McClements, D. J. (2003).
Production and characterization of oil-in-water emulsions containing
droplets stabilized by b-lactoglobulin–pectin membranes. Journal of
Agricultural and Food Chemistry, 51, 6612– 6617.
Netz, R. R., & Joanny, J.-F. (1999). Complexation between a semiflexible
polyelectrolyte and an oppositely charged sphere. Macromolecules, 32,
9026–9040.
Ogawa, S., Decker, E. A., & McClements, D. J. (2003a). Production and
characterization of O/W emulsions containing cationic droplets
stabilized by lecithin– chitosan membranes. Journal of Agricultural
and Food Chemistry, 51, 2806–2812.
Ogawa, S., Decker, E. A., & McClements, D. J. (2003b). Influence of
environmental conditions on stability of O/W emulsions containing
droplets stabilized by lecithin – chitosan membranes. Journal of
Agricultural and Food Chemistry, 51, 5522– 5527.
Ogawa, S., Decker, E. A., & McClements, D. J. (2004). Production and
characterization of O/W emulsions containing droplets stabilized by
lecithin–chitosan–pectin mutilayered membranes. Journal of Agricul-
tural and Food Chemistry, Vol. 52. pp. 3595–3600.
Pinotti, A., Bevilacqua, A., & Zaritzky, N. (1997). Optimization of the
flocculation stage in a model system of a food emulsion waste using
chitosan as polyelectrolyte. Journal of Food Engineering, 32, 69–81.
Pinotti, A., Bevilacqua, A., & Zaritzky, N. (1999). Treatment of anionic
emulsion systems using chitosan, polyacrylamide, and aluminum
sulfate. Scanning, 21, 354 –358.
Pinotti, A., Bevilacqua, A., & Zaritzky, N. (2001). Comparison of the
performance of chitosan and a cationic polyacrylamide as flocculants of
emulsion systems. Journal of Surfactants and Detergents, 4, 57–63.
Pinotti, A., & Zaritzky, N. (2001). Effect of aluminum sulfate and cationic
polyelectrolytes on the destabilization of emulsified wastes. Waste
Management, 21, 535–542.
Saito, H., Kawagishi, A., Tanaka, M., Tanimoto, T., Okada, S., Komatsu,
H., & Handa, T. (1999). Coalescence of lipid emulsions in floating and
freeze-thawing processes: Examination of the coalescence transition
state theory. Journal of Colloid and Interface Science, 219, 129 –134.
Schmitt, C., Sanchez, C., Desobry-Banon, S., & Hardy, J. (1998). Structure
and technofunctional properties of protein–polysaccharide complexes.
Critical Reviews in Food Science and Nutrition, 38, 689–753.
Shahidi, F., Arachchi, J. K. V., & Jeon, Y. J. (1999). Food applications of
chitin and chitosans. Trends in Food Science and Technology, 10,
37 –51.
Shi, X. Y., & Caruso, F. (2001). Release behavior of thin-walled
microcapsules composed of polyelectrolyte multilayers. Langmuir,
17, 2036–2042.
Skjak-Braek, G., Anthonsen, T., & Sandford, P. (1989). Chitin and
chitosan. London: Elsevier.
Stauffer, C. E. (1999). Emulsifiers. St Paul, MN: Eagen Press.
Strauss, G., & Hauser, H. (1986). Stabilization of lipid bilayer vesicles by
sucrose during freezing. Proceedings of the National Academy of
Science USA, 83, 2422–2426.
Vanapalli, S. A., Palanuwech, J., & Coupland, J. N. (2002). Stability of
emulsions to dispersed phase crystallization: Effect of oil type,
dispersed phase volume fraction, and cooling rate. Colloids and
Surfaces A, 204, 227–237.
Von Goeler, F., & Muthukumar, M. (1994). Adsorption of polyelectrolytes
onto curved spheres. Journal of Chemical Physics, 100, 7796–7803.
Walstra, P. (1993). Principles of emulsion formation. Chemical Engineer-
ing and Science, 48, 333–350.
Walstra, P (2003). Physical chemistry of foods. New York: Marcel Dekker.
Williams, P. A, & Phillips, G. O. (2003). The use of hydrocolloids to
improve food texture. In B. M. McKenna (Ed.), Texture in foods,
volume 1: Semi-solid foods, Boca Raton, FL: CRC Press, Chapter 11.